Target (protein/gene name): Inorganic pyrophosphatase *NCBI Gene # or RefSeq#:
ppa 3689032 *Protein ID (NP or XP #) or Wolbachia#: WP_004522531.1 *Organism (including strain): Burkholderia pseudomallei (strain: 1710b) Etiologic Risk Group (see link below):
Group 3 http://www.bacterio.net/-hazard.html */Disease Information (sort of like the Intro to your Mini Research Write up):
Melioidosis, or Whitmore's disease, is an infectious disease common in tropical climates caused by contaminated water and soil. There are several types of melioidosis infections, all with their own symptoms, including localized, pulmonary, bloodstream, and disseminated infections. Melioidosis's wide range of symptoms have often caused it to be mistake for other diseases such as pneumonia and tuberculosis. The disease is most prominent in Southeast Asia and Northern Australia.
"Pyrophosphatases catalyze the hydrolysis of inorganicphosphate (PPi) toinorganicphosphate Pi, driving biosynthetic reactions; they areessentialfor low cytosolicinorganicphosphate. It was suggested recently that posttranslational regulation of Family I solubleinorganicpyrophosphatases (sPPases) may affect their activity. As sPPases areessentialfor many metabolic pathways in eukaryotic cells"
"Inorganic pyrophosphatase (PPiase) is an essential enzyme that hydrolyzes inorganic pyrophosphate (PPi), driving numerous metabolic processes. We report a discovery of an allosteric inhibitor (2,4-bis(aziridin-1-yl)-6-(1-phenylpyrrol-2-yl)-s-triazine) of bacterial PPiases. Analogues of this lead compound were synthesized to target specifically Mycobacterium tuberculosis (Mtb) PPiase (MtPPiase). The best analogue (compound 16) with a Ki of 11 μM for MtPPiase is a species-specific inhibitor. Crystal structures of MtPPiase in complex with the lead compound and one of its analogues (compound 6) demonstrate that the inhibitors bind in a nonconserved interface between monomers of the hexameric MtPPiase in a yet unprecedented pairwise manner, while the remote conserved active site of the enzyme is occupied by a bound PPi substrate. Consistent with the structural studies, the kinetic analysis of the most potent inhibitor has indicated that it functions uncompetitively, by binding to the enzyme-substrate complex. The inhibitors appear to allosterically lock the active site in a closed state causing its dysfunctionalization and blocking the hydrolysis. These inhibitors are the first examples of allosteric, species-selective inhibitors of PPiases, serving as a proof-of-principle that PPiases can be selectively targeted."
*EC#: 3.6.1.1 Link to BRENDA EC# page: http://www.brenda-enzymes.org/enzyme.php?ecno=3.6.1.1 -- Show screenshot of BRENDA enzyme mechanism schematic Enzyme Assay information (spectrophotometric, coupled assay ?, reagents): -- link to Sigma (or other company) page for assay (see Sigma links below) http://www.sigmaaldrich.com/content/dam/sigma-aldrich/docs/Sigma/Enzyme_Assay/ionorgpyrophosph.pdf --- List cost and quantity of substrate reagents, supplier, and catalog #
Reagents:
Tris NaCl buffer
221368 Sodium pyrophosphate solution (pyrophosphate tetrasodium) 100g $33.60
M1028 Magnesium chloride solution 10X 1mL $44.60
A7302 Ammonium molybdate solution in H2SO4 100g $65.20
F7002 Tausky-Shorr Reagent (ammonium molybdate and ferrous sulfate) 250g $32.20
P3862 Phosphate standard solution 25mL $45.70
Expression Information (has it been expressed in bacterial cells): Yes Purification Method: http://www.jbc.org/content/241/9/1938.long Image of protein (PyMol with features delineated and shown separately): *Amino Acid Sequence (paste as text only - not as screenshot or as 'code'): MSFSNVPAGKDLPQDFNVIIEIPAQSEPVKYEADKALGLLVVDRFIGTGMRYPVNYGFIPQTLSGDGDPVDVLVITPFPLLAGSVVRARALGMLKMTDESGVDAKLVAVPHDKVCPMTANLKSIDDVPAYLKDQIKHFFEQYKALEKGKWVKVEGWDGIDAAHKEITDGVANFKK *length of your protein in Amino Acids 175 amino acids Molecular Weight of your protein in kiloDaltons using the Expasy ProtParam website 19161.14 Molar Extinction coefficient of your protein at 280 nm wavelength: 18450 TMpred graph Image (http://www.ch.embnet.org/software/TMPRED_form.html). Input your amino acid sequence to it.
results
*CDS Gene Sequence (paste as text only): GGCTATAATGCGCTTTCCATCGGAGGCGGCCGCGGGGCCGCCCTCCGCCCCGATTCCCATCCGAACTTCAGAGAACGCTCATGAGCTTCAGCAATGTTCCGGCCGGCAAGGATCTGCCGCAAGACTTCAACGTGATCATCGAGATCCCGGCGCAAAGCGAGCCGGTCAAGTACGAAGCCGACAAGGCGCTCGGCCTCCTCGTCGTCGACCGCTTCATCGGCACCGGCATGCGCTACCCGGTGAACTACGGCTTCATCCCGCAGACGCTGTCGGGCGACGGCGACCCCGTCGACGTACTCGTCATCACGCCGTTTCCGCTGCTCGCGGGCTCGGTCGTGCGCGCGCGCGCGCTCGGCATGCTGAAGATGACCGACGAATCGGGCGTCGACGCGAAGCTCGTCGCGGTGCCGCACGACAAGGTCTGCCCGATGACGGCCAACCTCAAGTCGATCGACGACGTGCCCGCGTACCTGAAGGATCAGATCAAGCACTTCTTCGAGCAATACAAGGCGCTCGAGAAGGGCAAGTGGGTGAAGGTCGAGGGCTGGGACGGCATCGACGCCGCGCACAAGGAAATCACCGACGGCGTCGCGAACTTCAAGAAGTAAGCCGCCGGGCGGCGCCCGCCGGGGCGCCCATGGGTCACGCGAAACCGCGCGCGCCTCGTCACGAGGCCGCGCGGTTTT (Inaccurate? 687... too high for 176 amino acids) - T.A.T. *GC% Content for gene: 65.6%
Do Not Need this info for Spring (but still copy these lines to your Target page for now)
*CDS Gene Sequence (codon optimized) - copy from output of Primer Design Protocol (paste as text only):
*GC% Content for gene (codon optimized):
Primer design results for pNIC-Bsa4 cloning (list seqeunces of all of your ~40 nt long primers): (link to DNA Works output text file - that should be saved in your Google Docs folder after you did the primer design protocol) -- Ask a mentor, Dr. B, or a fellow researcher -how to link a GDocs file if you are not sure how to. https://drive.google.com/open?id=0B4O2KqKh2q_-dGI1bVBqY2pXcEE
Primer design results for 'tail' primers (this is just 2 sequences): Forward Primer: 5’ TACTTCCAATCCATGTCTTTCTCTAACGTTCCA 3’ 33 bp
Reverse Primer: 5’ TATCCACCTTTACTGTCATTTTTTGAAGTTCGCAAC 3’ 36_ bp
Primers as designed by Todd: Forward Primer: 5' TACTTCCAATCCATGTCTTTCTCTAACGTTCCA 3' 33 bp 1.5 mM Mg++ 68.4 C Reverse Primer: 5' TATCCACCTTTACTGTCATTTTTTGAAGTTCGCA 3' 34 bp 1.5 mM Mg++ 68.4 C
*NCBI Gene # or RefSeq#:
ppa 3689032
*Protein ID (NP or XP #) or Wolbachia#:
WP_004522531.1
*Organism (including strain): Burkholderia pseudomallei (strain: 1710b)
Etiologic Risk Group (see link below):
Group 3 http://www.bacterio.net/-hazard.html
*/Disease Information (sort of like the Intro to your Mini Research Write up):
Melioidosis, or Whitmore's disease, is an infectious disease common in tropical climates caused by contaminated water and soil. There are several types of melioidosis infections, all with their own symptoms, including localized, pulmonary, bloodstream, and disseminated infections. Melioidosis's wide range of symptoms have often caused it to be mistake for other diseases such as pneumonia and tuberculosis. The disease is most prominent in Southeast Asia and Northern Australia.
Link to TDR Targets page (if present):
Link to Gene Database page (NCBI, EuPath databases -e.g. TryTryp, PlasmoDB, etc - or PATRIC, etc.)
https://www.ncbi.nlm.nih.gov/gene/3689032
Essentiality of this protein:
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3561365/
- "Pyrophosphatases catalyze the hydrolysis of inorganic phosphate (PPi) to inorganic phosphate Pi, driving biosynthetic reactions; they are essential for low cytosolic inorganic phosphate. It was suggested recently that posttranslational regulation of Family I soluble inorganic pyrophosphatases (sPPases) may affect their activity. As sPPases are essential for many metabolic pathways in eukaryotic cells"
Is it a monomer or multimer as biological unit? (make prediction at http://www.ebi.ac.uk/msd-srv/prot_int/pistart.html):6-mer
Complex of proteins?: No
Druggable Target (list number or cite evidence from a paper/database showing druggable in another organism):
https://www.ncbi.nlm.nih.gov/pubmed/27664315
*EC#: 3.6.1.1
Link to BRENDA EC# page:
http://www.brenda-enzymes.org/enzyme.php?ecno=3.6.1.1
-- Show screenshot of BRENDA enzyme mechanism schematic
Enzyme Assay information (spectrophotometric, coupled assay ?, reagents):
-- link to Sigma (or other company) page for assay (see Sigma links below)
http://www.sigmaaldrich.com/content/dam/sigma-aldrich/docs/Sigma/Enzyme_Assay/ionorgpyrophosph.pdf
--- List cost and quantity of substrate reagents, supplier, and catalog #
Reagents:
Tris NaCl buffer
221368 Sodium pyrophosphate solution (pyrophosphate tetrasodium) 100g $33.60
M1028 Magnesium chloride solution 10X 1mL $44.60
A7302 Ammonium molybdate solution in H2SO4 100g $65.20
F7002 Tausky-Shorr Reagent (ammonium molybdate and ferrous sulfate) 250g $32.20
P3862 Phosphate standard solution 25mL $45.70
Structure (PDB or Homology model)
http://www.rcsb.org/pdb/explore.do?structureId=3GVF
Current Inhibitors:
N-(pyridin-3-ylmethyl)aniline (DB06851)
5-amino-1-(4-chlorophenyl)-1H-pyrazole-4-carbonitrile (DB07291)
https://www.drugbank.ca/biodb/polypeptides/Q3JUV5
Expression Information (has it been expressed in bacterial cells): Yes
Purification Method:
http://www.jbc.org/content/241/9/1938.long
Image of protein (PyMol with features delineated and shown separately):
*Amino Acid Sequence (paste as text only - not as screenshot or as 'code'):
MSFSNVPAGKDLPQDFNVIIEIPAQSEPVKYEADKALGLLVVDRFIGTGMRYPVNYGFIPQTLSGDGDPVDVLVITPFPLLAGSVVRARALGMLKMTDESGVDAKLVAVPHDKVCPMTANLKSIDDVPAYLKDQIKHFFEQYKALEKGKWVKVEGWDGIDAAHKEITDGVANFKK
*length of your protein in Amino Acids
175 amino acids
Molecular Weight of your protein in kiloDaltons using the Expasy ProtParam website
19161.14
Molar Extinction coefficient of your protein at 280 nm wavelength:
18450
TMpred graph Image (http://www.ch.embnet.org/software/TMPRED_form.html). Input your amino acid sequence to it.
*CDS Gene Sequence (paste as text only):
GGCTATAATGCGCTTTCCATCGGAGGCGGCCGCGGGGCCGCCCTCCGCCCCGATTCCCATCCGAACTTCAGAGAACGCTCATGAGCTTCAGCAATGTTCCGGCCGGCAAGGATCTGCCGCAAGACTTCAACGTGATCATCGAGATCCCGGCGCAAAGCGAGCCGGTCAAGTACGAAGCCGACAAGGCGCTCGGCCTCCTCGTCGTCGACCGCTTCATCGGCACCGGCATGCGCTACCCGGTGAACTACGGCTTCATCCCGCAGACGCTGTCGGGCGACGGCGACCCCGTCGACGTACTCGTCATCACGCCGTTTCCGCTGCTCGCGGGCTCGGTCGTGCGCGCGCGCGCGCTCGGCATGCTGAAGATGACCGACGAATCGGGCGTCGACGCGAAGCTCGTCGCGGTGCCGCACGACAAGGTCTGCCCGATGACGGCCAACCTCAAGTCGATCGACGACGTGCCCGCGTACCTGAAGGATCAGATCAAGCACTTCTTCGAGCAATACAAGGCGCTCGAGAAGGGCAAGTGGGTGAAGGTCGAGGGCTGGGACGGCATCGACGCCGCGCACAAGGAAATCACCGACGGCGTCGCGAACTTCAAGAAGTAAGCCGCCGGGCGGCGCCCGCCGGGGCGCCCATGGGTCACGCGAAACCGCGCGCGCCTCGTCACGAGGCCGCGCGGTTTT
(Inaccurate? 687... too high for 176 amino acids) - T.A.T.
*GC% Content for gene: 65.6%
Do Not Need this info for Spring (but still copy these lines to your Target page for now)
*CDS Gene Sequence (codon optimized) - copy from output of Primer Design Protocol (paste as text only):
*GC% Content for gene (codon optimized):
Primer design results for pNIC-Bsa4 cloning (list seqeunces of all of your ~40 nt long primers):
(link to DNA Works output text file - that should be saved in your Google Docs folder after you did the primer design protocol)
-- Ask a mentor, Dr. B, or a fellow researcher -how to link a GDocs file if you are not sure how to.
https://drive.google.com/open?id=0B4O2KqKh2q_-dGI1bVBqY2pXcEE
Primer design results for 'tail' primers (this is just 2 sequences):
Forward Primer:
5’ TACTTCCAATCCATGTCTTTCTCTAACGTTCCA 3’ 33 bp
Reverse Primer:
5’ TATCCACCTTTACTGTCATTTTTTGAAGTTCGCAAC 3’ 36_ bp
Primers as designed by Todd:
Forward Primer: 5' TACTTCCAATCCATGTCTTTCTCTAACGTTCCA 3' 33 bp 1.5 mM Mg++ 68.4 C
Reverse Primer: 5' TATCCACCTTTACTGTCATTTTTTGAAGTTCGCA 3' 34 bp 1.5 mM Mg++ 68.4 C
**